Estimates of differential toxin expression governing heterogeneous intracellular lifespans of Streptococcus pneumoniae.

Following invasion of the host cell, pore-forming toxins secreted by pathogens compromise vacuole integrity and expose the microbe to diverse intracellular defence mechanisms. However, the quantitative correlation between toxin expression levels and consequent pore dynamics, fostering the intracellular life of pathogens, remains largely unexplored. In this study, using Streptococcus pneumoniae and its secreted pore-forming toxin pneumolysin (Ply) as a model system, we explored various facets of host-pathogen interactions in the host cytosol. Using time-lapse fluorescence imaging, we monitored pore formation dynamics and lifespans of different pneumococcal subpopulations inside host cells. Based on experimental histograms of various event timescales such as pore formation time, vacuolar death or cytosolic escape time and total degradation time, we developed a mathematical model based on first-passage processes that could correlate the event timescales to intravacuolar toxin accumulation. This allowed us to estimate Ply production rate, burst size and threshold Ply quantities that trigger these outcomes. Collectively, we present a general method that illustrates a correlation between toxin expression levels and pore dynamics, dictating intracellular lifespans of pathogens.

Galunisertib synergistically potentiates the doxorubicin-mediated antitumor effect and kickstarts the immune system against aggressive lymphoma.

In clinical practice, major efforts are underway to identify appropriate drug combinations to boost anticancer activity while suppressing unwanted adverse effects. In this regard, we evaluated the efficacy of combination treatment with the widely used chemotherapeutic drug doxorubicin along with the TGFßRI inhibitor galunisertib (LY2157299) in aggressive B-cell non-Hodgkin lymphoma (B-NHL). The antiproliferative effects of these drugs as single agents or in combination against several B-NHL cell lines and the synergism of the drug combination were evaluated by calculating the combination index. To understand the putative molecular mechanism of drug synergism, the TGF-ß and stress signaling pathways were analyzed after combination treatment. An aggressive lymphoma model was used to evaluate the anticancer activity and post-therapeutic immune response of the drug combination in vivo. Galunisertib sensitized various B-NHL cells to doxorubicin and in combination synergistically increased apoptosis. The antitumor activity of the drug combinations involved upregulation of p-P38 MAPK and inhibition of the TGF-ß/Smad2/3 and PI3K/AKT signaling pathways. Combined drug treatment significantly reduced tumor growth and enhanced survival, indicating that the synergism between galunisertib and Dox observed in vitro was most likely retained in vivo. Based on the tumor-draining lymph node analysis, combination therapy results in better prognosis, including disappearance of disease-exacerbating regulatory T cells and prevention of CD8+ T-cell exhaustion by downregulating MDSCs. Galunisertib synergistically potentiates the doxorubicin-mediated antitumor effect without aggravating the toxic effects and the ability to kickstart the immune system, supporting the clinical relevance of targeting TGF-ßRI in combination with doxorubicin against lymphoma.

Bioactive ZnO-assisted 1393 glass scaffold promotes osteogenic differentiation: Some studies.

We developed ZnO-assisted 1393 bioactive glass-based scaffold with suitable mechanical properties through foam replica technique and observed to be suitable for bone tissue engineering application. However, the developed scaffolds' ability to facilitate cellular infiltration and integration was further assessed through in vivo studies in suitable animal model. Herein, the pure 1393 bioactive glass (BG) and ZnO-assisted 1393 bioactive glass- (ZnBGs; 1, 2, 4 mol% ZnO substitution for SiO2 in pure BG is named as Z1BG, Z2BG, Z3BG, respectively) based scaffolds were prepared through sol-gel route, followed by foam replica techniques and characterized by a series of in vitro and some in vivo tests. Different cell lines like normal mouse embryonic cells (NIH/3T3), mouse bone marrow stromal cells (mBMSc), peripheral blood mononuclear cells, that is, lymphocytes and monocytes (PBMC) and U2OS (carcinogenic human osteosarcoma cells) were used in determination and comparative analysis of the biological compatibility of the BG and ZnBGs. Also, the alkaline phosphatase (ALP) activity, and osteogenic gene expression by primer-specific osteopontin (OPN), osteocalcin (OCN), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were performed to study osteogenic differentiability of the stromal cells in different BGs. Moreover, radiological and histopathological tests were performed in bone defect model of Wister rats to evaluate the in vivo bone regeneration and healing. Interestingly, these studies demonstrate augmented biological compatibility, and superior osteogenic differentiation in ZnBGs, in particular Z3BG than the pure BG in most cases.

γc cytokine-aided crosstalk between dendritic cells and natural killer cells together with doxorubicin induces a healer response in experimental lymphoma by downregulating FOXP3 and programmed cell death protein 1.

BACKGROUND AIMS: The stimulatory natural killer-dendritic cell axis in the tumor microenvironment could play a critical role in stimulating cytotoxic T cells and driving immune responses against cancer. METHODS: We established a novel treatment protocol by adroitly combining chemotherapy with doxorubicin and immunotherapy with dendritic cells and natural killer cells against a highly aggressive and malignant lymphoma called Dalton's lymphoma. RESULTS: Our data suggest that binary application of adoptive cell therapy and chemotherapy nearly cures (95%) early-stage experimental lymphoma. In the case of mid-stage cancer, the success rate was significantly lower but still impressive (75%). Our results demonstrated that the application of combination therapy in early-stage cancer significantly reduced the tumor volume and extended the lifespan of the experimental animal in addition to reinvigorating the immune system, including restoring the effector functions of dendritic cells and natural killer cells. The novel protocol limits the metastasis of tumor cells in vascularized organs and rearms the adaptive immune response mediated by dendritic cells and CD4+ and CD8+ T cells. CONCLUSIONS: Combination therapy in the early stage alters the cytokine profile, increases interferon-γ and tumor necrosis factor-α in the serum of treated animals and downregulates programmed cell death protein 1 expression in CD8+ T cells. Thus, cooperative and cognitive interactions between dendritic cells and natural killer cells in addition to therapy with doxorubicin promote the immune response and tumoricidal activities against lymphoma.

Targeting thymidine phosphorylase alleviates resistance to dendritic cell immunotherapy in colorectal cancer and promotes antitumor immunity.

T-cell exhaustion plays a pivotal role in the resistance of microsatellite-stable colorectal cancer (CRC) to immunotherapy. Identifying and targeting T-cell exhaustion-activating mechanisms is a promising strategy to augment the effects of immunotherapy. Here, we found that thymidine phosphorylase (TYMP) plays a decisive role in inducing systemic T-cell exhaustion and abrogating the efficacy of dendritic cell (DC) therapy in a CRC model. Targeting TYMP with tipiracil hydrochloride (TPI) induces immunological cell death (ICD). The combined effects of TPI and imiquimod-activated DCs turn CT26 tumors into immunologically 'hot' tumors by inducing ICD in vivo. High-dimensional cytometry analysis revealed T-cell and IFN-γ dependency on the therapeutic outcome. In addition, chemoimmunotherapy converts intratumoral Treg cells into Th1 effector cells and eliminates tumor-associated macrophages, resulting in higher cytotoxic T lymphocyte infiltration and activation. This effect is also associated with the downregulation of PD-L1 expression in tumors, leading to the prevention of T-cell exhaustion. Thus, cooperative and cognitive interactions between dendritic cells and immunogenic cell death induced by therapy with TPI promote the immune response and tumoricidal activities against microsatellite stable colorectal cancer. Our results support TYMP targeting to improve the effects of DC immunotherapy and outcomes in CRC.

PGC1 alpha coactivates ERG fusion to drive antioxidant target genes under metabolic stress.

The presence of ERG gene fusion; from developing prostatic intraepithelial neoplasia (PIN) lesions to hormone resistant high grade prostate cancer (PCa) dictates disease progression, altered androgen metabolism, proliferation and metastasis1-3. ERG driven transcriptional landscape may provide pro-tumorigenic cues in overcoming various strains like hypoxia, nutrient deprivation, inflammation and oxidative stress. However, insights on the androgen independent regulation and function of ERG during stress are limited. Here, we identify PGC1α as a coactivator of ERG fusion under various metabolic stress. Deacetylase SIRT1 is necessary for PGC1α-ERG interaction and function. We reveal that ERG drives the expression of antioxidant genes; SOD1 and TXN, benefitting PCa growth. We observe increased expression of these antioxidant genes in patients with high ERG expression correlates with poor survival. Inhibition of PGC1α-ERG axis driven transcriptional program results in apoptosis and reduction in PCa xenografts. Here we report a function of ERG under metabolic stress which warrants further studies as a therapeutic target for ERG fusion positive PCa.

Nanoscale Diamond-Based Formulation as an Immunomodulator and Potential Therapeutic for Lymphoma.

Integrative medicine practices, such as Ayurveda, are popular in India and many South Asian countries, yet basic research to investigate the concepts, procedures, and medical benefits of ayurvedic products has received little attention and is not fully understood. Here, we report a functional nanodiamond-based traditional Ayurvedic herbomineral formulation, Heerak Bhasma (Ayu_ND), for the treatment of solid tumors called Dalton's lymphoma generated in CD1 mice. Ayu_ND-mediated immunostimulation significantly reduces tumor cell proliferation and induces apoptosis aided by the active participation of dendritic cells. Immunomodulatory Ayu_ND treatment is highly immunostimulatory and drives dendritic cells to produce TNF-α. Treatment with Ayu_ND significantly reduces the tumor volume, inhibits metastasis in distant vascularized organs, and increases the life span of tumor-bearing animals compared with untreated littermates. These events were associated with elevated serum levels of the protective cytokines IFN-γ and TNF-α and downregulated the disease, exacerbating TGF-ß. Ayu_ND-mediated therapeutic success was also accompanied by the depletion of regulatory T cells and enhanced vaccine-induced T-cell immunity, guided by the restoration of the memory CD8+ T-cell pool and prevention of PD-1-mediated T cell exhaustion. The results provide a basis for further evaluation of ayurvedic formulations and drug efficacy in treating cancers.

Targeting PD-1 in CD8+ T Cells with a Biomimetic Bilirubin-5-fluoro-2-deoxyuridine-Bovine Serum Albumin Nanoconstruct for Effective Chemotherapy against Experimental Lymphoma.

We fabricated bilirubin-bovine serum albumin (BR-BSA) nanocomplexes as candidates for the delivery of 5-fluoro-2-deoxyuridine (5FUdr) against experimental murine lymphoma. BR was attached to 5FUdr via acid-labile ester bonds mimicking small-molecule drug conjugates. The construct was self-assembled with BSA through strong noncovalent interactions with high drug occupancy in the core and labeled with folic acid (FA) to target cancer cells. The BR-5FUdr-BSA-FA nanoconstruct exhibits excellent biocompatibility, prevents nephrotoxicity, and is tolerated by red blood cells and mononuclear cells. The construct also showed increased accumulation in lymph nodes and tumor cells. BR-5FUdr-BSA-FA caused prolonged growth inhibition and apoptosis, enhanced mitochondrial reactive oxygen species generation, and minimized the viability of parental and doxorubicin-resistant Dalton's lymphoma cells. Treatment of tumor-bearing mice with BR-5FUdr-BSA-FA significantly increased the life span of the animals, improved their histopathological parameters, and downregulated PD-1 expression, suggesting the potential of the construct for 5FUdr delivery to treat lymphoma.

Synthesis of drug conjugated magnetic nanocomposite with enhanced hypoglycemic effects.

In the present study, a magnetic nanocomposite (magnetite Fe3O4 and hematite Fe2O3) has been successfully synthesized by the sol-gel method and coated with polyvinyl alcohol (PVA) followed by conjugation of anti-diabetic drug metformin. Detailed structural and microstructural characterization of the nanocomposite (NP) and drug conjugated nanocomposite (NP-DC) are analyzed by the Rietveld refinement of respective XRD patterns, FTIR analysis, UV-Vis spectroscopy, SEM and TEM results. SEM and TEM image analyses reveal the spherical morphology and average size of NP, PVA coated nanoparticles (NP-PVA) and NP-DC samples, indicating a suitable size to be a nanocarrier. The biocompatibility of NP and NP-DC was carried out in NIH/3T3 and J774A. 1 cells. The enhanced activity of the drug, when conjugated with nanocomposite, is confirmed after the treatment of both the pure drug and NP-DC sample on the 18 h fasted normoglycemic and hyperglycemic mice. The blood glucose level of the mice is effectively decreased with the same concentration of the pure drug and NP-DC sample. It proves the increased activity of the NP-DC sample, as only 5 wt% drug is present that shows the same efficiency as the pure drug. This study suggests excellent biocompatibility and cytocompatibility of NP and NP-DC besides the critical property as a hypoglycemic agent. It is the first time approach of conjugating metformin with a magnetic nanocomposite for a significant increment of its hypoglycemic activity, which is very important to reduce the side effect of metformin for its prolonged use.

Galunisertib Drives Treg Fragility and Promotes Dendritic Cell-Mediated Immunity against Experimental Lymphoma.

Galunisertib (LY2157299) is a selective ATP-mimetic inhibitor of TGF-ß receptor-I activation, currently under clinical trial in a variety of cancers. We have tested the combined effects of galunisertib- and interleukin-15-activated dendritic cells in an aggressive and highly metastatic murine lymphoma. Based on the tumor-draining lymph node architecture, and its histology, the combination therapy results in better prognosis, including disappearance of the disease-exacerbating regulatory T cells. Our data suggest that galunisertib significantly enhances the success of immunotherapy with IL-15-activated dendritic cells by limiting the regulatory T cells generation with consequent downregulation of regulatory T cells in the tumor-draining lymph nodes and vascularized organ like spleen. This is also associated with consistent loss p-SMAD2 and downregulation of Neuropilin-1, leading to better prognosis and positive outcome. These results connect the role of combined therapy with the consequent elimination of disease-exacerbating T regulatory cells in a metastatic murine lymphoma.

Leishmanicidal Activity of an In Silico-Screened Novel Inhibitor against Ascorbate Peroxidase of Leishmania donovani.

Peroxidases are a heterogeneous family of enzymes that have diverse biological functions. Ascorbate peroxidase is a redox enzyme that is reduced by trypanothione, which plays a central role in the redox defense system of Leishmania In view of developing new and novel therapeutics, we performed in silico studies in order to search for a ligand library and identify new drug candidates and their physiological roles against promastigotes and intracellular amastigotes of Leishmania donovani Our results demonstrated that the selected inhibitor ZINC96021026 has significant antileishmanial effect and effectively killed both free and intracellular forms of the parasite. ZINC96021026 was found to be identical to ML-240, a selective inhibitor of valosin-containing protein (VCP), or p97, a member of the AAA-ATPase protein family which was derived from the scaffold of N2,N4-dibenzylquinazoline-2,4-diamine (DBeQ), targeting the D2-ATPase domain of the enzyme. ZINC96021026 (ML-240) thus has a broad range of cellular functions, thought to be derived from its ability to unfold proteins or disassemble protein complexes, besides inhibiting the ascorbate peroxidase activity. ML-240 may inhibit the parasite's ascorbate peroxidase, leading to extensive apoptosis and inducing generation of reactive oxygen species. Taken together, our results demonstrated that ML-240 could be an attractive therapeutic option for treatment against leishmaniasis.

A polynuclear Cu(ii) complex for real time monitoring of mitochondrial cytochrome C release during cellular apoptosis.

A new amide-imine conjugate, 2-hydroxybenzoic acid-(2-hydroxybenzylidene)-hydrazide (L1), is employed to prepare a single crystal X-ray structurally characterized poly-nuclear Cu(ii) complex (M1). M1 selectively and spatially interacts with cytochrome C (Cyt C) to allow fluorescence imaging of intracellular translocation events in living cells. Thus, direct visualization of a Cyt C translocation event during an apoptotic process is achieved for the first time. The binding constant and LOD are 7.52 × 104 M-1 and 34.0 nM, respectively.

Studies on interaction potency model based on drug synergy and therapeutic potential of triple stimuli-responsive delivery of doxorubicin and 5-fluoro-2-deoxyuridine against lymphoma using disulfide-bridged cysteine over mesoporous silica nanoparticles.

A triple stimuli-responsive drug delivery platform involving doxorubicin, 5-fluoro-2-deoxy uridine and folic acid was fabricated on mesoporous silica nanoparticles for targeting delivery against a highly aggressive murine lymphoma called Dalton's lymphoma. Fabrication of the unique construct by amalgamating active and passive targeting mechanisms offers a novel hyper-chimeric platform for a stimuli-responsive drug delivery system. The novel construct enables efficient and precise delivery of the precious cargo to the tumor sites. Active targeting by folic acid directs the doxorubicin and 5-fluoro-2-deoxy uridine in the close proximities of the tumor cells, causing efficient killing and significant growth inhibition. Isobologram models, zero interaction potency dose-response surface plots and matrices were generated to evaluate the combination synergism of the two drugs. Therapy with the dual drug-bearing construct in mice with established tumors significantly reduced the tumor load and enhanced the survival of the animals compared with the untreated control. Therapy with the dual delivery system also augmented the innate and adaptive immune defense mechanisms of the treated animals. CD8+ T cells, natural killer cells and the dendritic cells from the treated group following successful therapy with the novel construct showed enhanced cytotoxicity and growth inhibitory capacities against DL tumor cells.

Essential role of TNF-α in gamma c cytokine aided crosstalk between dendritic cells and natural killer cells in experimental murine lymphoma.

Cooperative and cognitive interaction between the dendritic cells and natural killer cells was investigated for demonstrating the anti-tumor activity against an aggressive murine lymphoma, treated with doxorubicin. Crosstalk between the dendritic cells and the natural killer cells significantly reduced the proliferation of Dalton's lymphoma cells in a dose dependent manner. Treatment of Dalton's lymphoma cells with doxorubicin in vitro enhances the effects of crosstalk against the target cells. This crosstalk between the cells was regulated via stimulation with recombinant interleukin-15, and release of TNF-α which is critically important for the tumoricidal effects. Dendritic cells and the natural killer cells crosstalk activate both the cells and upregulate the expression of CD40, CD69 and CD86 on the dendritic cells. These findings provided new insight regarding these interactions and define a mechanism by which cellular immune response promotes tumoricidal activity against lymphoma in therapeutic setting.

In silico studies and evaluation of antiparasitic role of a novel pyruvate phosphate dikinase inhibitor in Leishmania donovani infected macrophages.

The present work deals with the identification and characterization of a novel inhibitor Z220582104, specific to pyruvate phosphate dikinase, for leishmanicidal activities against free promastigotes and intracellular amastigotes. We have used structure-based drug designing approaches and performed homology modelling, virtual screening and molecular dynamics studies. Primary mouse macrophages and macrophage cell line J774A1 were infected with promastigotes of Leishmania donovani. Both promastigotes and infected macrophages were subjected to treatment with the varying concentrations of Z220582104 or miltefosine for assessment of leishmanicidal activity. The novel inhibitor Z220582104 demonstrated growth inhibitory potential and reduced the viability of the free promastigotes in a concentration- and time-dependent manner. Z220582104 was also effective against the intracellular form of the parasites and reduced the number of amastigotes in macrophages and also lowered the parasite index, compared with the untreated infected macrophages. Although less effective compared with the miltefosine, Z220582104 is well tolerated by the dividing cells and normal human lymphocytes and monocytes with no adverse effects on the growth kinetics or viability. Our in silico and in vitro studies suggested that Leishmania donovani pyruvate phosphate dikinase could be a potential new drug target.